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First published online 12 November 2008
doi: 10.1242/dev.028209
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MRC Centre for Developmental Neurobiology, New Hunt's House, Guy's Campus, King's College London, SE1 1UL, UK.
e-mail: julian.ng{at}kcl.ac.uk
Accepted 15 October 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural development, Signal transduction, Cytoskeleton, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Multiple aspects
of axonal development are regulated by Rho GTPases. Although Rac generally
mediates axon extension and attractive responses, and Rho1 (also known as
RhoA) generally mediates axon retraction and repulsion, these distinctions can
be complex. For example, Drosophila genetic studies show axon
outgrowth and attractive responses mediated by Netrin
(Forsthoefel et al., 2005),
and axon repulsive cues mediated by Robo
(Fan et al., 2003;
Matsuura et al., 2004), both
of which depend on Rac subfamily GTPases. Similarly, Drosophila Rho1
signals can mediate axon retraction
(Billuart et al., 2001) and
attraction (Bashaw et al.,
2001) in different neurons. These and many other studies highlight
the key and complex roles that Rho GTPases play in growth cone responses.
Studies on mushroom body (MB) neurons in the Drosophila brain have
shown that Rho proteins regulate axon growth through LIM kinase
(LIMK)-dependent and -independent pathways, and that they can act
antagonistically (Ng and Luo,
2004). LIMK regulates actin filament turnover by phosphorylating,
and thereby inactivating, an actin depolymerisation and severing factor,
ADF/cofilin (Bamburg, 1999).
LIMK1 misexpression in neurons, in vitro or in vivo, leads to axon growth
inhibition. Consistent with a role in ADF/cofilin regulation, this phenotype
is suppressed by increasing cofilin activity, either by coexpressing wild-type
cofilin or a form (S3A) that cannot be phosphorylated, or by expressing the
cofilin phosphatase, Slingshot (Ssh) (Endo
et al., 2003; Ng and Luo,
2004). In Drosophila, one homologue of ADF/cofilin
exists, twinstar (tsr), and its inactivation results in
growth cone morphology and axon growth defects. These results suggest that
cofilin phosphoregulation is essential for axon growth.
How extracellular cues pattern axons through Rho GTPase and cofilin
regulation in vivo is unclear. Here I show that components of the Transforming
growth factor beta (TGFβ) pathway are involved. The TGFβ pathway
regulates many morphogenic events, including cell fate specification, cell
migration, proliferation and apoptosis
(Hogan, 1996;
Massague et al., 2000;
Raftery and Sutherland, 1999).
The conserved TGFβ pathway consists of a core complex of type 1 and type
2 transmembrane receptor serine/threonine kinases, which are activated by
secreted TGFβ ligands [bone morphogenetic proteins (BMPs) or
TGFβ/Activins] (Feng and Derynck,
2005; Shi and Massague,
2003). The presence of ligand dimers triggers a signalling cascade
involving the receptor complex. The following events are essential:
phosphorylation of type 1 receptors by the type 2 receptor kinase;
phosphorylation of receptor activated Smads (R-Smads) by the type 1 receptor
kinase; R-Smad complex formation with a common Smad (co-Smad); translocation
of Smad complexes into the nucleus to elicit gene transcription. In
Drosophila, there are three type 1 receptors, Baboon (Babo),
Thickveins (Tkv) and Saxophone (Sax), and two type 2 receptors, Wishful
thinking (Wit) and Punt (Put). The activated receptors phosphorylate two
R-Smads, Mad and Smad2 (also known as dSmad2 and Smox - FlyBase), which form a
trimeric complex with the co-Smad Medea (Med). In most models, Smad activation
is an obligate effector response upon ligand binding.
Although Smad-independent pathways are known
(Derynck and Zhang, 2003;
Moustakas and Heldin, 2005;
Foletta et al., 2003;
Lee-Hoeflich et al., 2004;
Ozdamar et al., 2005), how
they affect development in vivo is unclear. In many instances,
Smad-independent pathways exhibit cross-regulatory effects, which either
regulate Smads or are under Smad regulation. However, some TGFβ signals
are Smad-independent events. In C. elegans, mutations in a TGFβ
signal (unc-129) result in dorsal-ventral axon guidance defects
(Colavita et al., 1998).
Mutation analyses of other TGFβ components, such as receptors or Smads,
do not reveal this phenotype, suggesting that axon guidance in worms involves
atypical TGFβ signalling mechanisms. TGFβ signals also regulate
dorsal-ventral axon guidance in the developing mouse spinal cord. BMP7
expression in the dorsal roof plate acts to repel spinal cord neurons and
guide their projections ventrally
(Augsburger et al., 1999;
Butler and Dodd, 2003). Whether
Smads are involved is unclear; nonetheless, the rapid axonal responses would
seem to preclude transcriptional events.
Recent studies have shown that BMP4 and BMP7 treatment in mammalian
non-neuronal and neuronal cell cultures, respectively, leads to LIMK
activation, resulting in a rapid increase in cofilin phosphorylation
(Foletta et al., 2003;
Lee-Hoeflich et al., 2004).
This requires a direct interaction between the C-terminal tail of a BMP
receptor (BMPR2), which is dispensable for Smad signalling, and LIMK.
Lee-Hoeflich et al. (Hoeflich et al., 2004) have further shown that the BMPR2
C-terminus is required for dendritogenesis in cultured cortical neurons.
Mammalian BMPs also regulate growth cone turning responses in cultured
Xenopus spinal neurons (Wen et
al., 2007). BMP7 exposure causes attractive or repulsive growth
cone turning behaviours by regulating cofilin through LIMK1 or Ssh activities,
respectively.
Drosophila LIMK1 is essential for synaptic stability controlled by
BMPs. Genetic analysis of the Drosophila neuromuscular junction (NMJ)
reveals that the stability of presynaptic terminals requires a retrograde
BMP-type signal, Glass bottom boat (Gbb), that acts through Wit (the
Drosophila homologue of BMPR2). Like BMPR2, Wit binds to LIMK1 via
its C-terminal extension. Without this interaction, NMJ synapses can grow
(through Wit signalling via the Drosophila Smads, Mad and Medea) but
they have defects in synaptic stability
(Eaton and Davis, 2005). How
TGFβ receptor interactions regulate LIMK1 is unclear
(Foletta et al., 2003;
Lee-Hoeflich et al., 2004).
Nor is it clear how LIMK1 regulates synapses, as cofilin phosphoregulation
does not appear to be essential (Eaton and
Davis, 2005).
Here, I show that TGFβ signals regulate distinct aspects of axonal development. Loss of Babo results in MB axon overextension, whereas in other neurons axon outgrowth and targeting defects are observed. The results show that Babo acts together with Wit and Put, but is independent of Smads. Babo signals depend on Rho1, Rac and LIMK1. Consistent with a role in LIMK1 regulation, babo and wit genetically interact with LIMK1. babo and LIMK1 gain-of-function phenotypes are similar, and both are suppressed by increasing cofilin activity. Contrary to the canonical receptor activation model, the type 2 receptors Wit and Put both act downstream of the Babo type 1 receptor, and distinct LIMK1-dependent and -independent pathways are required.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The following additional strains were used:
babo32, babo52, UAS-activated baboa
Q302D (CA babo) (Brummel et al.,
1999); tkv4, tkv7
(Penton et al., 1994);
tkv4a21 (Gibson and
Perrimon, 2005); UAS-put
I,
UAS-tkv1GSK (DN tkv),
UAS-saxI (DN sax),
UAS-tkv1A (HA) Q199D (CA tkv), UAS-saxA
(HA) Q263D (CA sax) (Haerry et
al., 1998); sax4
(Singer et al., 1997);
saxP, UAS-put (Nellen
et al., 1994); UAS-babo-a, UAS-babo-b::Flag,
UAS-baboaI (DN babo)
(Zheng et al., 2006) (a gift
from M. O'Connor, HHMI/University of Minnesota, Minneapolis and Theo Haerry,
Florida Atlantic University, Boca Raton); witA12,
witB11, witG15, P{wit genomic}
(P{wit+}), P{wit tailless}
(P{witC}), UAS-wit, UAS-witC
(Marques et al., 2002);
UAS-witI (McCabe
et al., 2003); put135, UAS-put
(Ruberte et al., 1995);
put62 (Simin et al.,
1998); Mad12
(Sekelsky et al., 1995);
Med13 (Hudson et al.,
1998); Smad21
(Zheng et al., 2003);
UAS-Dad (Tsuneizumi et al.,
1997); UAS-MYC::tum (RacGAP50C)
(Goldstein et al., 2005);
UAS-EcR-B1 (Lee et al.,
2000); Df(1)HF368, UAS-RhoGEF2 (Bloomington
Drosophila Stock Center). Constitutively active (CA) forms of type 1
receptors result from a conserved Gln (Q) to Glu (D) mutation leading to
constitutively active kinase activity
(Wieser et al., 1995).
Dominant-negative (DN) forms of type 1 and type 2 receptors derive from
cytoplasmic deletions, with the loss of intracellular domains (cited above).
Genetic crossing schemes used in this study are available upon request.
MARCM and Gal4-UAS expression studies
Loss-of-function clones were generated using the MARCM method
(Lee and Luo, 1999).
Neuroblast and single-cell 
β clones were generated as previously
described (Ng et al., 2002).
Neurons were visualised using the Gal4-OK107 driver expressing
UAS-mCD8::GFP. Misexpression studies were performed using the same
driver. For CA and DN misexpression studies, unless indicated otherwise,
multiple copies (2-4) of the UAS transgene were used to derive the strongest
possible phenotypes. The strength of CA Babo phenotypes was correlated with
Babo expression levels, using one, two or four copies of UAS-CA babo
(data not shown; Figs 4,
5,
7 and see Fig. S2 and Fig. S6D
in the supplementary material). The data shown in Figs
4,
5 and
7 were obtained using two
copies (UAS lines 1B and 9B). MARCM clones were visualised by immunostaining
using anti-CD8 (Caltag, clone CT-CD8a, 1:100) and anti-Fas2 (a gift from G.
Tear, King's College London; clone 1D4, 1:5) antibodies. In misexpression
studies, neurons were visualised using epifluorescent CD8::GFP together with
anti-Fas2 staining. Additional antibodies used were HA (Santa Cruz, Y11,
1:500), Babo (Abcam, ab14681, 1:50), Wit (a gift from H. Aberle, MPI
Developmental Biology, Tübingen; clone 23C7, 1:10) and FLAG (Sigma, clone
M5, 1:200). These were used to estimate the level and localisation of ectopic
Sax-HA, Tkv-HA, Babo, Wit and WitC-FLAG proteins, respectively, in
neurons. Although endogenous Babo and Wit were detected throughout brain
tissue, ectopic levels were distinguished using these antibodies.
Drosophila brains were dissected, fixed and stained as previously
described (Ng et al., 2002).
Confocal images were generated with a Zeiss LSM510 confocal microscope, using
Zeiss LSM510, Image J and Adobe Photoshop software.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kurusu et al.,
2002; Lee et al.,
1999). There are three different sets of adult MB neurons
(
, 'β' and β), which are born at
different periods from common neuroblast progenitors and have distinct axonal
projections (Lee et al., 1999)
(Fig. 1A,B). Each neuron
extends a primary neurite that gives rise to dendrites near the cell body, and
a single axon that projects anteroventrally through the peduncle. Axons of
'β' or β neurons bifurcate to form a
dorsal and a medial branch, whereas neurons extend only a medial
branch (branches are also referred to collectively as `lobes'). All axons
terminate either medially, close to the midline, or close to the anterior
dorsal cortex (Fig. 1A,B).
Babo inactivation results in MB axon overextension
To study the role of TGFβ signals in MB neurons, mutant clones were
generated using strong loss-of-function or null alleles of the type 1
receptors babo, tkv and sax. babo-null
(babo52) neuroblast clones had axon overextension
phenotypes in β neurons, with β lobes overextending across
the midline (Fig. 1, compare C
with B, quantified in H). Consistent with previous studies
(Zheng et al., 2003),
babo clones also exhibited axon pruning defects, characterised by the
presence of larval-stage dorsal and medial projections in adult brains (open
white arrowheads in Fig. 1C).
In wild-type adults, each neuron re-extends a single medial branch
after axon pruning and the lobe appears more defasciculated along the
dorsal-ventral axis (Fig. 1B).
By mutant clonal analysis or by dominant-negative (DN) misexpression, loss of
tkv or sax did not result in these defects
(Fig. 1D,E,H; data not shown).
These results suggest that Babo regulates axon growth, particularly of the
β lobe.
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). Expression of the Baboa, but not
Babob, isoform rescues the babo MB axon pruning
phenotypes. By contrast, either isoform rescues the babo axon
extension defects of dorsal cluster (DC) neurons in the optic lobe. To test
whether different Babo isoforms regulate MB axon growth, similar assays were
performed. In a wild-type background, ectopically expressed Baboa
or Babob was detected in all MB lobes and did not disrupt axonal
projections (see Fig. S2 in the supplementary material). Baboa or
Babob expression in babo52 neuroblast clones
rescued the axon overextension defect, as most β lobes terminated
correctly (Fig. 1F-H). Thus,
either Babo isoform can regulate axon growth.
Consistent with a cell-autonomous role, Babo inactivation in single
β neurons resulted in similar axon overextensions. Interestingly,
non-cell-autonomy was also observed, as single babo neurons caused
heterozygote axons to similarly overextend across the midline (see Fig. S1 in
the supplementary material).
Babo regulates MB axon growth independently of axon pruning
Using a different approach, a DN form of Babo was misexpressed in MB
neurons. Like the null phenotype, axon pruning and overextension phenotypes
were observed, with β lobes fusing at the midline
(Fig. 2A,A'; 65.2% fusion
defects, n=23 brains). To determine whether axon overextension was
secondary to axon pruning defects, DN babo was misexpressed together
with the Ecdysone receptor B1 isoform (EcR-B1). Similar to previous
results (Zheng et al., 2003),
these axon pruning defects were suppressed by ectopic EcR-B1
(Fig. 2B'). However,
β lobe fusions remained visible (64.5%, n=31;
Fig. 2B). Therefore, DN Babo
axon overextension was not secondary to the axon pruning defects. Conversely,
nor were axon pruning defects a consequence of axon overextension, as
UAS-babob expression rescued
babo52 axon overextension but not the axon pruning defects
(Fig. 1G). Similarly, RhoGEF2
coexpression also suppressed DN Babo axon overextension but not the axon
pruning defects (Fig.
2C,C'; see below). Thus, Babo regulates axon pruning and
axon growth independently.
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; Das et al.,
1999; Zheng et al.,
2003). When Smad2 strong loss-of-function clones were
analysed, axon overextension defects were not detected, although, consistent
with previous data (Zheng et al.,
2003), axon pruning defects were
(Fig. 3A, quantified in
Fig. 1H). Null clones of
Medea (Med, the Drosophila homologue of the co-Smad
Smad4) also did not exhibit overextension defects
(Fig. 3B and
Fig. 1H). Recent data suggest
that, under certain in vitro conditions, Babo can signal through Mad
(Gesualdi and Haerry, 2007).
When Mad-null, or Smad2 Mad double mutant clones were
analysed, axon overextensions were not observed either (data not shown;
Fig. 3C and
Fig. 1H). Similarly, in a
different strategy, misexpression of an inhibitory form of Smad, Dad,
also did not perturb these axons (data not shown; 100% as wild-type,
n=26 hemispheres). As Smads could play a redundant role, their role
was tested in a sensitised background. Using a Babo gain-of-function
phenotype, one mutant copy of either Smad2, Mad or UAS-Dad
was introduced with constitutively active (CA) babo
(Fig. 5A,B; see below).
Reducing Smad levels did not suppress CA Babo. In fact, loss of Mad,
or Dad misexpression, enhanced CA Babo phenotypes. Together, these
results suggest that Babo regulates axon growth independently of Smads.
Expression of constitutively active Babo inhibits axon growth
To determine how Babo functions independently of Smads, a gain-of-function
approach was taken. CA forms of type 1 receptors were misexpressed in MB
neurons. CA Babo expression resulted in axon truncation phenotypes, with the
loss of dorsal and/or medial branches (Fig.
4A,A'; for quantification see
Fig. 5). Axon guidance defects
were also observed; however, this phenotype represented a small fraction of
animals [classed as misguidance (MG) in Figs
5,
7; see Fig. S2A,B in the
supplementary material]. To test whether CA Babo phenotypes were simply due to
increased levels of Babo protein, ectopic wild-type Babo levels were compared
with CA Babo levels (see Fig. S2 in the supplementary material). The results
showed that the dominant CA Babo phenotype is due to the Q302D mutation, which
results in higher kinase activity. High levels of CA Tkv and CA Sax protein
were detected in MB axons (data not shown). Nevertheless, these axon
projections resembled those of the wild type (CA tkv, 100% as
wild-type, n=26 hemispheres; CA sax, 92.1% as wild-type,
n=38 hemispheres; Fig.
4B,C). These results again suggest that Babo, but not Tkv or Sax,
regulates axon growth in vivo.
To determine whether the truncation phenotypes reflect an initial failure of axon extension, as opposed to axons failing to stabilise and subsequently retracting, CA babo-misexpressing animals were developmentally staged and analysed from wandering L3 larvae (data not shown) through to puparium formation. The results suggest that CA Babo resulted in early extension defects in developing axons (see Fig. S3 in the supplementary material).
babo and wit genetically interact with LIMK1
LIMK1 misexpression results in similar MB axon phenotypes to those
described above (Fig. 4,
compare D with A) (Ng and Luo,
2004). However, in contrast to LIMK1, which also led to
lobe truncations, only β lobes were truncated in CA
babo-misexpressing animals. Additionally, in CA babo, β
lobes were predominantly disrupted (Fig.
4A'; see quantification in
Fig. 5A,B).
To study the link between TGFβ and LIMK1, receptor mutants were introduced to determine whether they could modify the LIMK1 misexpression phenotype (Fig. 4E). Loss of one copy of babo or wit suppressed the LIMK1 phenotype. LIMK1 misexpression was not suppressed by other type 1 receptors, such as tkv or sax, or by the other type 2 receptor, put. These genetic assays suggest that Babo and Wit positively interact with LIMK1.
Babo-regulated axon growth requires components of the Rho1 and Rac pathway
Drosophila LIMK1 is regulated by Rho GTPases (Rho1, Rac and Cdc42)
through the effector kinases, Rok and Pak
(Ng and Luo, 2004). To
determine whether Babo-regulated axon growth requires the Rho GTPase pathway,
genetic interaction assays were performed using CA babo
(Fig. 5A). Lowering the level
of Rho1 signals, by loss of one copy of Rho1 or of the Rho1 activator
RhoGEF2, resulted in suppression of the CA Babo phenotype. Loss of
the Rho1 effector kinase, Rok, also suppressed CA Babo.
When other Rho family members, Cdc42 and Rac (Rac1,
Rac2 or Mtl), were tested, loss of Rac1 (using the
hypomorphic allele J10), or a combined loss of one copy of
Rac2 and Mtl (using null alleles), also suppressed
CA Babo (Fig. 5A). Stronger
allelic combinations of Rac enhanced the CA Babo phenotypes
(unpublished observations). This is expected, based on previous observations
that Rac GTPases can play opposite roles in promoting and inhibiting MB axon
growth (Ng and Luo, 2004;
Ng et al., 2002). Loss of
Cdc42 did not suppress CA Babo. Loss of the Cdc42/Rac effector kinase
Pak also did not suppress CA Babo, but instead resulted in stronger
CA Babo phenotypes. These results suggest that in addition to Rho1, CA
Babo-mediated axon growth inhibition also requires Rac, but not Cdc42 or
Pak.
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Whether inhibiting Rho pathways through RhoGAPs affects CA Babo was then
tested (Fig. 5B). In a
wild-type background, single-copy expression of UAS-RhoGAPp190 or
UAS-tumbleweed (tum, also known as RacGAP50C) did
not disrupt normal axonal projections, although, as previously described,
RhoGAPp190 caused a mild dorsal lobe overgrowth defect
(Billuart et al., 2001;
Goldstein et al., 2005).
RhoGAPp190, which acts as a Rho1 inhibitor, strongly suppressed CA Babo
(Fig. 5B; data not shown). This
is consistent with previous findings that ectopic RhoGAPp190 also suppresses
LIMK1 misexpression phenotypes (Ng and
Luo, 2004). Tum expression also suppressed CA Babo
(Fig. 5B; data not shown).
Drosophila tum genetically interacts with Rac1 in the wing
and eye (Sotillos and Campuzano,
2000) and tum mutant clones exhibit MB axon extension
defects (Goldstein et al.,
2005).
Together, this suggests that Babo-regulated axon growth requires the Rho1 and Rac GTPases and involves RhoGEFs (RhoGEF2) and RhoGAPs (RhoGAPp190 and Tum) (Fig. 7E; see below).
DN Babo-induced axon overextension is suppressed by increased Rho1 activity
Based on these results, one would predict that DN Babo-induced axon
overextension (Fig. 2A; 65.2%
fusion defects, n=23 brains) would be suppressed by increased Rho1
signals. Thus, when RhoGEF2 was coexpressed with DN Babo, axon overextension
was suppressed (Fig. 2C; 8.7%
fusion defects, n=46 brains). RhoGEF2 did not affect the DN Babo axon
pruning defect (Fig.
2C'). Similarly, Rok coexpression also suppressed DN Babo
axon overextension, but not the axon pruning phenotype (11.8%, n=34;
data not shown).
Other RhoGEFs were tested, but none of these suppressed the DN babo-induced axon overextensions (UAS-pbl, 51.9%, n=77; UAS-trio, 63.3%, n=60; UAS-sif, 43.9%, n=41; data not shown). Taken together, these results suggest that Babo-regulated axon growth requires Rho1 through the activator RhoGEF2 and the effector kinase Rok (Fig. 7E).
CA Babo is suppressed by loss of LIMK1 and by increased cofilin activity
Given their similar phenotypes, the link between CA Babo and LIMK1 was
analysed further. Loss of one copy of LIMK1 [using the deficiency
Df(1)HF368] strongly suppressed the CA babo axon truncation phenotype
(Fig. 5A). Intriguingly, β
lobe overextensions were observed in many CA babo brains (15 out of
17 brains; see Fig. S4 in the supplementary material), suggesting that CA Babo
promotes axon extension under low LIMK1 levels. As the LIMK1 misexpression
phenotype is inhibited by Drosophila cofilin (Tsr)
(Ng and Luo, 2004),
tsr was coexpressed with CA babo. Consistent with its
predicted role in regulating LIMK1, Tsr (tsr WT) expression
suppressed CA Babo (data not shown; Fig.
5B). However, the results suggest that Babo does not regulate
cofilin phosphorylation alone (see Discussion).
Type 2 receptors Wit and Put regulate axon growth independently and interchangeably
Whether TGFβ type 2 receptors regulate axon growth was tested.
wit-null neuroblast clones exhibited β lobe overextensions
similar to those of babo mutants
(Fig. 6A,G, compare with
Fig. 1C). Since the Wit
C-terminal tail binds to LIMK1 (Eaton and
Davis, 2005), the relevance of this region was analysed.
Consistent with previous results, wit mutants are viable in the
presence of the `tailless' genomic rescue transgene
(P{witC}), which lacks the Wit C-terminal region but
includes the kinase region (Marques et
al., 2002) (data not shown). However, compared with the wild-type
full-length wit genomic construct (P{wit+}), the
tailless wit transgene failed to suppress the wit-null
overextensions (data not shown; Fig.
6G). This suggests that the C-terminal region is essential for
Wit-regulated axon growth.
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I) was misexpressed
(Fig. 6C; 45.5% fusion defects,
n=44 brains).
To test whether type 2 receptors can function interchangeably,
UAS-put was expressed in wit clones. wit axon
overextensions were suppressed by Put expression
(Fig. 6D,G). Conversely,
put phenotypes were rescued by UAS-put or UAS-wit
(data not shown; Fig. 6E,G).
However, put phenotypes were not rescued by the tailless
UAS-witC (Fig.
6F,G). These results suggest that although Wit and Put regulate
axon growth independently, they can function interchangeably. However,
distinct mechanisms are employed, involving LIMK1-dependent and -independent
pathways (Fig. 7E) (see
Discussion).
The type 2 receptors Wit and Put act downstream of the type 1 receptor Babo
The results suggest that Babo, Wit and Put work together. In the canonical
model of TGFβ signalling, type 1 receptors act downstream of type 2
receptors. Furthermore, activated type 1 receptors propagate Smad signals
independently of ligands or type 2 receptors
(Brummel et al., 1999;
Wieser et al., 1995) and, in
vivo, result in ectopic TGFβ responses independently of ligands
(Haerry et al., 1998;
Lecuit et al., 1996;
Nellen et al., 1996). Using CA
Babo, the relevance of this model was tested
(Fig. 7A). Loss of one copy of
wit suppressed CA Babo. Expression of a DN form of wit
(UAS-witI), which alone did not disrupt MB axon
projection (data not shown), also suppressed CA Babo. In similar assays, one
mutant copy of put, or UAS-putI
coexpression, also suppressed CA Babo. These results suggest that Babo
regulates axon growth together with Wit and Put. However, contrary to the
canonical model, CA Babo requires the presence of type 2 receptors.
To explore this further, genetic epistasis experiments were performed. Wit and Put were expressed in babo-null neurons (Fig. 7B,C, quantified in D). Ectopic Wit or Put suppressed the babo axon overextension but not the axon pruning phenotype (a Smad-dependent process). Collectively, these results suggest that in Babo-regulated axon growth, type 2 receptors act downstream of type 1 signals (Fig. 7E).
Babo regulates axon extension and targeting of AL and OL axons independently of Smads
To determine whether Babo regulates the axon patterning of other neurons,
antennal lobe (AL) and optic lobe (OL) contralateral projection neurons were
analysed (Ng and Luo, 2004)
(Fig. 8A,B,F). As previously
described, these neurons extend axons contralaterally into the opposite AL
(Fig. 8A,B) or OL
(Fig. 8A,F), respectively.
babo AL and OL clones showed axonal defects
(Fig. 8C,G, quantified in J).
babo AL axons were disrupted in the target area and fewer axons
extended across the midline. babo OL axons displayed a subtler
phenotype: although the number of babo OL axons projecting into the
initial target area appeared normal, terminal branches were less elaborated
and `gaps' were observed in terminal zones (open blue arrowheads in
Fig. 8G; see Fig. S5 in the
supplementary material). No gross misprojections were observed. These results
suggest that Babo regulates axon extension and targeting in AL neurons, but
only axon targeting in OL neurons.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Role of Smad-independent signals in neural connectivity
Once growing axons reach the correct postsynaptic target, axon outgrowth
terminates and synaptogenesis begins. These studies suggest that TGFβ
signals play a role. When Babo is inactivated, MB axon growth does not
terminate properly and overextends across the midline. Consistent with this,
CA Babo expression results in precocious termination, forming axon
truncations. How Babo is spatially and temporally regulated remains to be
determined. Analogous to the Drosophila NMJ, MB axon growth might be
terminated through retrograde signalling. Target-derived TGFβ ligands
could signal to Babo (on MB axon growth cones) and stop axons growing further.
In an alternative scenario, TGFβ ligands might act as a positional cue
that prevents MB axons from crossing the midline. Recent data have shown that
Babo acting through Smad2 restricts individual R7 photoreceptor axons to
single termini (Ting et al.,
2007). Loss of Babo, Smad2, or the nuclear import regulator
Importin 3 (Karyopherin 3 - FlyBase), results in R7 mutant axons
invading neighbouring R7 terminal zones. With the phenotype described here,
Babo could similarly be restricting MB axons to appropriate termination zones,
its loss resulting in inappropriate terminations on the contralateral
side.
In contrast to MB neurons, Babo inactivation in AL and OL neurons resulted
in axon extension and targeting defects. This might reflect cell-intrinsic
differences in the response in different neurons to a common Babo signalling
programme. This may be the case for MB axon pruning and DC axon extension,
which require Babo/Smad2 signals (Zheng et
al., 2006). Whether these differences derive from cell-intrinsic
properties, or from Babo signal transduction, they underline the importance of
Smad-independent signals in many aspects of axonal development.
Role of Rho GTPases in TGFβ signalling
The results suggest that Smad-independent signals involve Rho GTPases. One
caveat in genetic interaction experiments is that the loss of any given gene
might not be dosage-sensitive with a particular assay. Nevertheless, all the
manipulations together suggest that Babo-regulated axon growth requires Rho1,
Rac and LIMK1. How Babo signals involve Rho GTPases remains to be fully
determined. In addition to LIMK1, which binds to Wit, one possibility, as
demonstrated for many axon guidance receptors
(Luo, 2002), is that the
RhoGEFs, RhoGAPs and Rho proteins might be linked to the Babo receptor
complex. Thus, ligand-mediated changes in receptor properties would lead to
spatiotemporal changes in Rho GTPase and LIMK1 activities.
The data suggest that a RhoGEF2/Rho1/Rok/LIMK1 pathway mediates Babo responses (Fig. 7E). Whether Rac activators are required is unclear, as tested RacGEFs do not genetically interact with babo. In this respect, rather than through GEFs, Babo might regulate Rac through GAPs, by inhibiting Tum activity (Fig. 7E).
Do mutations in Rho1 and Rac components phenocopy babo phenotypes?
β lobe overextensions are observed in Rok
(Billuart et al., 2001),
Rho1 and Rac mutant neurons (unpublished observations). In
MB neurons, Rac GTPases also control axon outgrowth, guidance and branching
(Ng et al., 2002). Rho1 also
has additional roles in MB neurons
(Billuart et al., 2001).
Although Rho1 mutant neuroblasts have cell proliferation defects,
single-cell β clones do show β lobe extensions (unpublished
observations). RhoGEF2 strong loss-of-function clones do not exhibit
axon overextension (unpublished observations). As there are 23 RhoGEFs in the
Drosophila genome (Adams et al.,
2000; Hu et al.,
2005), there might well be redundancy in the way Rho1 is
activated. LIMK1 inactivation in MB neurons was reported previously
(Ng and Luo, 2004). Axon
overextensions were not observed as LIMK1 loss results in axon outgrowth and
misguidance phenotypes. This suggests that LIMK1 mediates multiple axon
guidance signals, of which TGFβ is a subset in MB morphogenesis.
|
), CA babo is dosage-sensitive only to Rho1
and Rac and specific Rho regulators (this study), suggesting that
Babo regulates LIMK1 only through a subset of Rho signals.
Second, the LIMK1 misexpression phenotype is suppressed by
expression of wild-type cofilin (Tsr), S3A Tsr, or the cofilin phosphatase Ssh
(Ng and Luo, 2004). By
contrast, only wild-type Tsr, but not S3A Tsr or Ssh
(Fig. 5B; unpublished
observations), suppresses CA Babo. The suppression by wild-type Tsr might
reflect a restoration of the endogenous balance or spatial distribution of
cofilin-on (unphosphorylated) and -off (phosphorylated) states within neurons.
Indeed, optimal axon outgrowth requires cofilin to undergo cycles of
phosphorylation and dephosphorylation
(Meberg and Bamburg, 2000;
Ng and Luo, 2004). As S3A
forms of cofilin cannot be inactivated and recycled from actin-bound
complexes, wild-type cofilin is more potent in actin cytoskeletal
regulation.
CA Babo might not simply misregulate LIMK1 but also additional cofilin
regulators. Recent data suggest that extracellular cues (including mammalian
BMPs) can regulate cofilin through Ssh phosphatase
(Endo et al., 2007;
Nishita et al., 2005;
Wen et al., 2007) and
phospholipase C activities
(Mouneimne et al., 2006;
van Rheenen et al., 2007). In
different cell types, cofilin phosphorylation and phospholipid binding (which
also inhibits cofilin activity) states vary and potently affect cell motility
and cytoskeletal regulation. Whether a combination of LIMK1, Ssh and
phospholipid regulation affects cofilin-dependent axon growth remains to be
determined.
Third, by phalloidin staining, LIMK1, but not CA Babo, misexpression results in a dramatic increase in F-actin in MB neurons (see Fig. S6 in the supplementary material). Thus, CA Babo does not in itself lead to actin misregulation. Fourth, Babo also regulates axon growth independently of LIMK1 (see below).
Role of Babo, Wit and Put in neuronal morphogenesis
This study differs significantly from the canonical model of Smad
signalling (Feng and Derynck,
2005; Shi and Massague,
2003), in which type 1 receptors function downstream of the
ligand-type 2 receptor complex (Wieser et
al., 1995). In this study, the gain- and loss-of-function results
suggest that type 2 receptors act downstream of type 1 signals. As ectopic Wit
and Put only suppress the babo axon overextension phenotype, this
implies that Smad-dependent and -independent signals have distinct type 1/type
2 receptor interactions. How these interactions propagate Smad-independent
signals remains to be fully determined. Babo could act as a ligand-binding
co-receptor with Wit and Put. In addition, Babo kinase activity could regulate
type 2 receptor or Rho functions. The results suggest, however, that provided
that Wit or Put signals are sufficiently high, Babo is not required. Whatever
the mechanism(s), it is likely that Babo requires the Wit C-terminus-LIMK1
interaction to relay cofilin phosphoregulatory signals
(Fig. 7E). How Put functions is
unclear. As the put135 allele (used in this study) carries
a missense mutation within the kinase domain, this suggests that kinase
activity is essential. put does not genetically interact with
LIMK1. As Put lacks the C-terminal extension of Wit that is necessary
for LIMK1 binding, this suggests that Put acts independently of LIMK1. One
potential effector is Rac, which, in the context of Babo signalling, also
appears to be Pak1- and thus LIMK1-independent
(Fig. 7E).
|
).
However, as the Wit C-terminal tail is required to substitute for Put, this
suggests that Wit axon growth signals are independent of its kinase activity.
Together, this suggests that Smad-independent signals involve LIMK1-dependent
and -independent mechanisms.
Distinct roles of Babo in neuronal morphogenesis
This study, together with Zheng et al.
(Zheng et al., 2003), shows
that Babo mediates two distinct responses in related MB populations. How do MB
neurons choose between axon pruning and axon growth? The babo rescue
studies suggest that whereas Baboa or Babob elicits
Smad-independent responses, only Baboa mediates Smad-dependent
responses. As Babo isoforms differ only in the extracellular domain,
differences in ligand binding could determine Smad2 or Rho GTPase activation.
However, it is worth noting that in DC neurons, either isoform mediates axon
extension through Smad2 and Medea (Zheng
et al., 2006). In addition, although expressed in all MB neurons,
CA babo misexpression (which confers ligand-independent signals)
perturbs only β axons (Fig.
4A,A' and see Fig. S2 in the supplementary material). Thus,
cell-intrinsic properties might also be essential in determining Babo
responses.
Many TGFβ ligands signal through Babo
(Gesualdi and Haerry, 2007;
Lee-Hoeflich et al., 2005;
Parker et al., 2006;
Serpe and O'Connor, 2006;
Zheng et al., 2003;
Zhu et al., 2008). For
example, Dawdle, an Activin-related ligand, patterns Drosophila motor
axons (Parker et al., 2006;
Serpe and O'Connor, 2006),
whereas Activin (Activin-β, FlyBase) is required for MB axon pruning
(Zheng et al., 2003). Whether
these ligands regulate Babo MB, AL and OL axonal morphogenesis is unclear.
Taken together, the evidence suggests that Babo signalling is varied in vivo
and is involved in many aspects of neuronal development.
Smad-independent signals in cytoskeletal regulation and cell morphogenesis
TGFβ signals are responsible for many aspects of development and
disease and, throughout different models, Smad pathways are closely involved.
Although Smad-independent pathways are known, their mechanisms and roles in
vivo are unclear. TGFβ signals often drive cell shape changes in vivo.
During epithelial-to-mesenchymal transition (EMT), cells lose their epithelial
structure and adopt a fibroblast-like structure that is essential for cell
migration during development and tumour invasion
(Grunert et al., 2003;
Shook and Keller, 2003).
TGFβ-mediated changes in the actin cytoskeleton and adherens junctions
are necessary for EMT. Although Smads are crucial, TGFβ signals also
involve the Cdc42-Par6 complex, resulting in cell de-adhesion and F-actin
breakdown through Rho1 degradation
(Ozdamar et al., 2005). In
other studies, however, TGFβ-mediated EMT has been shown to require Rho1
(Bhowmick et al., 2001), which
can be regulated by Smad activity (Levy
and Hill, 2005).
Many TGFβ-driven events in Drosophila are Smad-dependent
(Raftery and Sutherland,
1999). Whether Smad-independent roles exist beyond those
identified in this study remains to be tested. Here, I provide a framework to
understand how non-Smad signals regulate cell morphogenesis during
development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/4025/DC1
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ACKNOWLEDGMENTS |
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