1 Department of Genetics, Cell Biology and Development, Howard Hughes Medical
Institute, University of Minnesota, Minneapolis, MN55455, USA
2 The University of Texas at Austin, Section of Neurobiology Institute for
Cellular and Molecular Biology, 227 Patterson Laboratories, Austin, TX78712,
USA
¶ Author for correspondence (e-mail: moconnor{at}mail.med.umn.edu)
Accepted 29 July 2003
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SUMMARY |
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Key words: FMRFamide, Neurohemal organ, Neuromuscular junction
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Introduction |
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The physiological processes that FaRPs influence are quite varied. The
original invertebrate peptide was isolated from clam ganglia and exhibited
marked cardioexcitatory activity (Price
and Greenberg, 1977). Other family members, such as Myosuppressins
and Sulfakinins, affect spontaneous contractions of the visceral and oviduct
muscles and thereby regulate feeding and egg-laying behaviors
(Lange and Orchard, 1998
;
Nachman et al., 1986
;
Wang et al., 1994
;
Wang et al., 1995a
;
Wang et al., 1995b
). In
Aplysia, these peptides can influence learning and memory
(Guan et al., 2002
;
Mackey et al., 1987
;
Small et al., 1989
) while in
vertebrates endogenous FaRPs regulate analgesic effects of opiate peptides and
influence the electrical activity of some central brain synapses
(Askwith et al., 2000
;
Gayton, 1982
;
Kavaliers, 1990
;
Kavaliers and Yang, 1991
;
Nishimura et al., 2000
;
Tang et al., 1984
;
Yang et al., 1985
).
In invertebrates, FaRPs can also influence body wall muscle activity. Early
studies in the locust suggested that FMRF-like peptides enhance synaptic
transmission at the neuromuscular junction
(Robb and Evans, 1994), and
more recent studies in crustaceans suggest that these peptides modulate
presynaptic Ca2+-channel activity
(Rathmayer et al., 2002
). In
Drosophila, peptides produced from the FMRFa gene can also
enhance synaptic efficacy at the neuromuscular junction when perfused onto
standard larval nerve-muscle preparations
(Hewes et al., 1998
). Such
treatment results in a significant increase in muscle contraction or twitch
tension. Drosophila FMRFa is expressed in many neurosecretory cells,
including the Tv neurons that innervate a specialized tissue known as the
neurohemal organ (NHO). As the NHO releases products into the hemolymph, FaRPs
probably act systemically in a hormone-like fashion to regulate NMJ synaptic
activity in vivo (O'Brien et al.,
1991
; Schneider et al.,
1991
; Schneider et al.,
1993a
; Schneider et al.,
1993b
). Elucidating the mechanisms that control the peptidergic
phenotype and activity of particular neurosecretory cells is therefore
important for understanding how NMJ activity may be modulated by the
neuroendocrine system.
Within the vertebrate nervous system, members of the bone morphogenetic
protein (Bmp) subgroup of TGFß and their cognate receptors have been
implicated in controlling several different aspects of neural development and
function, including neurulation, morphogenesis, lineage decisions and cellular
maturation (reviewed by Mehler et al.,
1997). Bmps have also been implicated in neural specification
processes. For example, the induction and maintenance of the neuronal
cholinergic phenotype in the central nervous system is influenced by Bmp9
(Lopez-Coviella et al., 2000
),
and trunk neural crest cells are induced to an adrenergic phenotype by Bmp2,
Bmp4 and Bmp7 (Reissmann et al.,
1996
; Varley and Maxwell,
1996
). Other Bmps have also been implicated in regulating
neurotransmitter expression in sympathetic
(Fann and Patterson, 1994
;
Lo et al., 1998
;
Schneider et al., 1999
),
spinal cord (Kalyani et al.,
1998
), mesencephalic (Jordan
et al., 1997
; Reiriz et al.,
1999
), striatal (Hattori et
al., 1999
) and serotonergic neurons
(Galter et al., 1999
). In most
of these cases, the Bmp is not required for the differentiation of these
neurons, rather it helps them obtain their final phenotypic characteristics by
inducing the expression of genes specific for the function of that neuron. In
C. elegans, daf-7, a TGFß-type ligand, is required for
maintaining, but not initiating, the expression of chemoreceptors in sensory
neurons, thus modulating the chemosensory properties of specific neurons
(Nolan et al., 2002
).
With these examples as precedent, we sought to determine whether Bmp
signals might influence the expression of neuroendocrine phenotypes in
Drosophila. We and others have recently described a novel
Drosophila Bmp type 2 receptor, coded for by the wishful
thinking (wit) locus, that is primarily expressed in, and
required for, proper nervous system function
(Aberle et al., 2002;
Marqués et al., 2002
).
Mutations in wit result in pharate lethality caused, in part, by
defects in the growth and physiology of motoneuron synapses. We show here that
mutations in wit also affect the peptidergic phenotype of certain
FMRFa-expressing cells. In particular, we find that FMRFa
expression is eliminated in the Tv neurons that contribute to the systemic
supply of FMRFa peptides through release at the neurohemal organ. We show that
the regulation of FMRFa expression in Tv neurons is mediated by the
Bmp ligand Gbb, as gbb null mutations also eliminate FMRFa
expression in Tv neurons. Furthermore, we demonstrate that supplying Gbb to
the dorsal neurohemal cells restores FMRFa expression in Tv neurons.
As Tv neuron axons arborize onto the neurohemal cells, this strongly suggests
that Gbb signals in a retrograde manner to specify the peptidergic phenotype
of Tv neurons. Consistent with this view, we find that overexpression in
neuroendocrine cells of Dynamitin or a dominant-negative form of p150/Glued,
both components of the Dynactin/Dynein motor complex, also eliminates FMRFa
expression in the Tv neurons. Finally, we show that providing FMRFa
in neuroendocrine cells using the Gal4/UAS system partially rescues the lethal
phenotype of wit mutants, even though they still exhibit structural
and physiological synaptic defects. We suggest that Bmp signaling provides a
global cue that not only regulates the growth of the NMJ synapses locally
(Aberle et al., 2002
;
Marqués et al., 2002
;
McCabe et al., 2003
) but also
controls their systemic modulation by the neuroendocrine system.
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Materials and methods |
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Construction of UAS>FMRFa
UAS>FMRFa was generated by synthesis of two 100 bp complementary
oligonucleotides that encode the last few amino acids of the FMRFa
gene. After formation of duplexes, this oligo was inserted into the
PstI-SpeI sites of an incomplete FMRFa cDNA
(Schneider et al., 1991) to
generate a full-length coding region. The full-length FMRFa gene was
excised from the pBluescript clone as an EcoRI-SpeI fragment
and inserted into the EcoRI-XbaI sites of pUAST
(Brand and Perrimon, 1993
).
Construction of chimeric receptors
pUC2
pCasper-Ubiquitin (pUC) (Brummel et al.,
1994) was digested with EcoRI, blunted with Klenow, and
religated. The plasmid was cut with XbaI and StuI, and a new
polylinker was added. Unique sites in the polylinker: 5' NotI,
SacII, HpaI, AvrII, EcoRI, XbaI
3'.
pUC-sax (StuI)
A StuI site (AGGCCT) was inserted into the middle of the sax
transmembrane domain in pBluescript by site-directed mutagenesis. A
NotI fragment was inserted into pUC.
pUC-tkv1 (StuI)
A StuI site (AGGCCT) was inserted into the middle of the
transmembrane domain of the tkv1 isoform in pBluescript by site-directed
mutagenesis. A NotI fragment was inserted into pUC.
pUC2-wit5' (StuI, HpaI)
StuI (AGGCCT) and HpaI (GTTAAC) sites were inserted into
the middle of the wit transmembrane domain in pBluescript by site-directed
mutagenesis. A HindIII (blunted)-HpaI fragment was inserted
into the HpaI site of pUC2.
pUC2-wit (StuI, HpaI)
A StuI-XbaI fragment of pBluecript-wit5'
(StuI, HpaI) was replaced from pBluecript-wit5'
(StuI, HpaI)
Chimeric receptors
The chimeric proteins are fused in the middle of the transmembrane
domains.
Antibody staining
The following antibodies were used at the indicated dilutions for
characterization of the wit and gbb mutant phenotypes:
monoclonal anti-lacZ (Promega) 1/1000; rabbit anti-PSMAD1 (ten
Dijke), 1/1000 in embryos and 1/500 in larvae; monoclonal anti-Csp (Zinsmaier)
1/400; and rabbit anti-FMRFamide (Peninsula Laboratories) 1/1000. The Alexa
series (Molecular Probes) of secondary antibodies were used for
immunofluorescence at 1/500 dilution. Larvae were dissected and fixed in 4%
formaldehyde in PBS containing 0.5 mM EGTA, 5 mM MgCl2 for 10-20
minutes at room temperature for pMad and lacZ staining. For FMRFamide
staining, a protocol was used in which fixation takes place in 0.1 M
NaPO4, 0.3% Triton X-100, 0.1%sodium azide, 0.1% BSA for 2-6 hours
(McCormick et al., 1999).
Staining of all tissues was visualized in a Zeiss Axioplan2 with a CARV unit
for confocal microscopy.
Electrophysiology
The standard third instar larval body-wall muscle preparation was used for
electrophysiology as previously described
(Jan and Jan, 1976;
Zhang et al., 1998
). Briefly,
excitatory junctional potentials (EJPs) in muscles 6 and 7 were elicited by
stimulating the innervating motor nerve bundle with a suction electrode in
HL-3 solution containing 1 mM CaCl2. The recording microelectrode
had an input resistance between 15 M
and 25 M
. EJPs were
acquired and digitized using a PC computer with the use of pCLAMP 8 software
(Axon Instruments). The analysis and presentation of figures were conducted on
Clampfit (Axon Instruments) and Origin (Origin lab). Samples used for final
analysis were obtained from at least five different larvae. ANOVA and Unpaired
Student's t-test were used for data treatment (mean±s.e.). The
resting potential of the muscles was between 65.8±1.2 mV for w
control, 64.9±0.6mV for witA12/D(3)C175, and
63.9±1.6 mV for the FRMFa rescue. They were not statistically
different from each other (P>0.5).
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Results |
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Wit is required in Tv neurons to activate expression of
FMRFa
As wit expression is observed in many neuronal cells, we wished to
determine if it is required directly in Tv neurons or whether it might
function indirectly via an interneuron signal. To address this issue, we
employed three different Gal4 lines that express Gal4 in either all or
specific subsets of neuronal cells. The elav driver is expressed in
most differentiated neurons and, supplying wit with this driver
restores FaRP expression in the Tv neurons of wit mutants
(Fig. 2A). The C929 and 386
lines express Gal4 in much more limited sets of neuroendocrine cells
(Taghert et al., 2001),
including the Tv neurons but not the NHO (for C929 see
Fig. 2C-F). As these drivers
also rescue FaRP accumulation in the Tv neurons and the NHO varicosities (2B),
we conclude that wit is required only in the Tv neurons and is not
required to mediate an interneuron signal, nor is it required in NHO cells. In
order to confirm that Wit is required in Tv neurons, we took advantage of the
variable expression of the OK6 driver in Tv neurons. We have noticed that in
addition to the previously described motoneuron expression (Aberle, 2002),
this driver is also expressed in a random fashion in some or all of the Tv
neurons. We looked at FMRFa expression in wit mutants
rescued with a witGFP transgene driven by OK6>Gal4. We find that
FMRFa expression is variably recovered in different animals, but in
all cases (five animals, 17 Tv neurons) the expression of FMRFa
correlates with the expression of WitGFP in those neurons
(Fig. 3). In these experiments,
we could also detect WitGFP in the NHO (not shown). In no case did we see
WitGFP expression in Tv neurons without FMRFa expression.
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Although these findings demonstrate that reception of a Bmp signal is an essential component of FMRFa gene regulation in Tv neurons, they do not rule out that an activin-type signal may also be received and required by the Tv neurons to stimulate FMRFa expression. To probe this issue, we examined FMRFa expression in baboon mutant third instar larvae. As shown in Fig. 5G, we found that expression of the FMRFa/lacZ transgene is still evident in babo mutant larvae, demonstrating that an activin-type signal is not required for FMRFa expression.
Retrograde Gbb signaling is required for FaRP expression in Tv
neurons
The Drosophila genome contains seven TGFß type ligands. Three
of these, Dpp, Screw and Gbb, have been shown to transduce Bmp-type signals
(Mad) and to use the type I receptors Tkv and Sax
(Brummel et al., 1994;
Haerry et al., 1998
;
Nguyen et al., 1998
). Two
others, Activin and Activin-like protein, transduce signals through Smad2
(T.E.H. and M.B.O., unpublished). The signaling pathways used by Maverick and
Myoglianin remain untested. Among the three Bmp-type ligands, Gbb seemed a
likely candidate for controlling expression of FMRFa as it is broadly
expressed, at least in embryos (Doctor et
al., 1992
; Wharton et al.,
1991
), and can signal through Wit to regulate P-Mad accumulation
in motoneurons and tissue culture cells
(McCabe et al., 2003
). As
shown in Fig. 6A, gbb
is strongly expressed in the larval brain lobes and much more weakly in the
ventral ganglia. Interestingly, we also note that gbb shows enriched
expression in the NHO relative to other ventral ganglia neurons
(Fig. 6A). Thus, Gbb is
expressed in the correct place to be a FMRFa regulating ligand.
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The fact that gbb is expressed in the NHO onto which the Tv neuron axons terminate suggested that perhaps Gbb signals to these neurons via a retrograde mechanism. To address this issue, we took advantage of the observation that the 24B>Gal4 driver, while expressed in muscles, is also specifically expressed in the NHO cells (Fig. 7D-F) but not in the Tv neurons themselves (Fig. 7A-C). When 24B>Gal4 is used to express Gbb in the NHO, we found that FaRP expression is restored in the Tv neurons (Fig. 7G), while as shown above, use of the muscle specific driver G14>Gal4 does not rescue (Fig. 6F).
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To demonstrate that these effects are not the result of interference with
P-Mad transport from the cell cytoplasm to the nucleus, we also expressed
Gl in the embryonic gut using the Y45>Gal4 driver
(Wharton et al., 1999
). We
have previously shown that P-Mad accumulates extensively in the midgut
beginning at stage 15 of embryogenesis
(Marqués et al., 2002
).
Overexpression of
Gl did not interfere with P-Mad accumulation in the
midgut (Fig. 8D versus 8E), nor
did it interfere with P-Mad accumulation in dorsal cells when a ubiquitous
Gal4 driver such as daughterless is used
(Fig. 8F versus 8G). The only
tissue showing a loss of P-Mad staining when using the ubiquitous
da>Gal4 line is the CNS
(McCabe et al., 2003
). We
conclude that P-Mad accumulation in neurons is most sensitive to disruption of
Dynein motor function, consistent with the notion that Bmp signaling in these
cells requires retrograde transport of at least one component as part of its
signal transduction mechanism.
Overexpression of FMRFa partially rescues wit mutants
As FaRPs are known to enhance synaptic transmission at the neuromuscular
junction, we wished to determine if re-supplying FMRFa alone could
partially ameliorate the wit mutant phenotype. We have previously
shown that wit mutants exhibit structural defects at active zones and
are defective in synaptic transmission
(Aberle et al., 2002;
Marqués et al., 2002
).
Our rationale was that, although re-supply of FaRPs would probably not rescue
the primary structural defect of the synapses, it might enhance synaptic
transmission enough to allow some animals to eclose. For this purpose we
constructed a UAS>FMRFa transgene and expressed it in
neuroendocrine cells using C929>Gal4. At 25°C we find that
80%
(n=65) of wit mutant pharate adults can partially escape
from the pupal case (Fig. 9B).
In the absence of FMRFa expression less than 0.5% of wit
mutants (0 of 212) open the operculum. At 28°C about 30% of the females
can fully eclose (Fig. 9C) and
some inflate their wings (Fig.
9D). While these animals move fairly well they usually die within
several days without producing progeny. Although many of these animals are
able to eclose, we see no rescue in the size of the synapse
(Fig. 9F,G) nor is the primary
defect in synaptic transmission rescued
(Fig. 9H,I). We conclude
therefore, that the inability of wit mutant animals to eclose is the
result of at least two defects, one in NMJ synaptic growth and
neurotransmitter release, and the other in the production of systemic FaRPs
that act as neuromodulators at the NMJ.
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Discussion |
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In Drosophila FMRFamide peptides have been shown to enhance
synaptic transmission and muscle twitch tension when perfused onto standard
larval nerve-muscle preparations (Hewes et
al., 1998); however, their in vivo role(s) are not known as no
mutations in the FMRFa gene have been identified. As with most
neuropeptides, FaRPs are thought to act as neuromodulators and neurohormones.
The Tv-produced FaRPs are released into the hemolymph through the neurohemal
organ and hence are able to act on every tissue in the animal that is not
blocked to hemalymph contact. We have previously hypothesized that the
lethality of wit mutants is due to the lack of proper synaptic
transmission at the NMJ, resulting in the animals not being able to eclose
from the pupal case (Marqués et
al., 2002
). The lack of systemic FMRFamide described here would be
expected to further decrease synaptic efficiency and the ability of
wit mutants to eclose. The fact that loss of FMRFa does contribute to
the lethal phenotype is supported by the partial rescue of wit
mutants by overexpression of FMRFa. These results are consistent with
the view that in vivo, FMRFa peptides probably enhance NMJ synaptic activity
similar to their in vitro documented effects on standard larval
electrophysiological preparations.
It is important to note that although the lethal phenotype is partially reversed, the morphological and physiological synaptic defects reported for wit mutants are not rescued by overexpression of FMRFa. The simplest interpretation is that the excess of FaRPs enhances the efficiency of wit mutant synapses in vivo without correcting the underlying developmental defects. Although one might expect a significant improvement of the electrophysiological phenotype, this is not detected (Fig. 9), probably because the excess FaRPs are either washed off the preparation during standard dissection prior to recording or act for only short periods.
Although we have argued that the FMRF rescue effect is the result of
enhance synaptic transmission at the NMJ, we can not exclude the possibility
that the rescue of eclosion by FMRFa expression in wit
mutants is due to a central effect on ecdysis regulation, and not to a
peripheral effect on the NMJ. The driver used, C929, expresses FMRFa
in all neurosecretory cells, so extra FMRFamide is conceivably produced in the
CNS. However, it is important to recognize that wit mutants are
defective only in the systemic, Tv-secreted form of FMRFamide. Although
FMRFamide has a well-studied effect in modulating visceral and somatic muscle
contraction (Nichols, 2003),
no effect for FMRFamide in triggering eclosion has been uncovered
(Fuse and Truman, 2002
;
Zitnan et al., 2003
). In
fact, FaRPs accumulate in a model of developmental arrest in Manduca
(Zitnan et al., 1995
). Even
if overexpression of FMRFa in all neurosecretory cells in the
wit rescue experiment could be due to a novel role for FMRFamide in
ecdysis regulation in the CNS, it seems unlikely that this would apply in the
wild-type condition, in which the Wit pathway is only regulating systemic
FMRFamide. For the systemic peptide to have any central effect it would have
to cross the blood-brain barrier that sheaths the nervous system
(Carlson et al., 2000
;
Kretzschmar and Pflugfelder,
2002
). Although this is possible, it seems more likely that the
systemic delivery system has specifically evolved to function only on
peripheral tissues and not the CNS itself. Effects of FMRFs in the CNS seem
more likely to be mediated by the other neuroendocrine cells that are not
affected in wit mutants. It is apparent that a complete dissection of
the roles of systemic FMRFamide verse central FMRFs in synaptic transmission
and eclosion will need to await the isolation of FMRFa mutants.
How Wit signaling regulates FMRFa expression is not clear. As
Smads are well known to act as transcriptional co-activators or co-repressors
(Attisano and Wrana, 2002;
Moustakas et al., 2001
) the
simplest explanation is that Mad directly regulates activation of
FMRFa transcription, perhaps by forming a complex with Ap. However,
other indirect mechanisms are also possible and this issue will only be
resolved once the FMRFa promoter is fully characterized. It is also
not clear whether Gbb is the only ligand that regulates FMRFa
expression through Wit. In some developmental contexts, such as wing imaginal
disc patterning, Gbb acts in combination with Dpp, another Bmp-type ligand
(Haerry et al., 1998
;
Khalsa et al., 1998
). We do
not see any expression of dpp in the NHO. However, it could be that
one of the as yet uncharacterized ligands, Maverick or Myoglianin, could be a
partner with Gbb in regulating FMRFa expression. Conversely, it seems
clear that regulating the peptidergic phenotype of the six Tv neurons is not
the only role of Gbb signaling. There are hundreds of neurons that receive Bmp
signaling as indicated by P-Mad nuclear localization Most of them appear to be
motoneurons, which require Wit/Gbb signaling to achieve proper synaptic growth
but not to specify their neurotransmitter phenotype (Aberle, 2002;
Marqués, 2002). Given that Smads act as co-transcriptional regulators,
the fact that the same signal (nuclear translocation of P-Mad) results in
different phenotypic outcomes in different neurons can probably be ascribed to
the presence of a different set of transcription factors available in each
cell type. The Tv neurons receiving the Bmp signal express apterous,
a transcription factor required in those cells for FMRFa
transcription (Benveniste, 1998), and maybe other factors that are required,
in addition to the Wit signal, to activate FMRFa.
Another important issue to resolve is whether Gbb is constitutively
released from the NHO, or is synthesized and released as part of a feedback
mechanism to modulate muscle contractions. It might be that efficient muscle
contraction under normal conditions requires a constant level of FaRPs that
are produced in response to a constitutive Gbb signal. Alternatively, Gbb
production or release might be regulated by a sensing mechanism that would
activate the pathway in response to an increased demand for FaRPs, owing to
increased locomotor activity or other stimuli, such as compensating for a
synaptic developmental defect. We have recently found that muscle-derived Gbb
acts through neuronal Wit to convey a retrograde signal essential for NMJ
synapse growth and maturation (McCabe et
al., 2003). In that context, it appears that the role of Bmp
signaling is to coordinate muscle growth with synapse maturation to ensure
proper synaptic efficiency. Thus, our combined results indicate that the
Wit/Gbb pathway acts as a two-step regulator of NMJ function. First, there is
a developmental role in which Wit signaling is required for proper synaptic
growth during larval development (Aberle et
al., 2002
; Marqués, 2002;
McCabe et al., 2003
). Second,
Wit signaling is required to achieve the neuromodulatory effect of circulating
FaRPs that are required for optimal synaptic transmission. Lack of either one
of these inputs probably results in a substantial decrease of the EJCs. These
two examples suggest that the Gbb/Wit pathway is of general importance in
neural retrograde signaling and we speculate that it may be used in the
nervous system for other as yet uncharacterized developmental and
physiological purposes.
Retrograde signaling by target-derived Gbb
Tv neurosecretory cells form part of a cluster of four
apterous-expressing neurons on each side of the three thoracic
ganglia. The axons of the Tv neurons extend proximally and dorsally to join
the contralateral axon, and form a median nerve that swells and arborizes onto
a group of neurons and glial cells that constitute the neurohemal organ
(Benveniste et al., 1998). In
wit mutants, these structures develop normally, but the Tv neuron
fail to activate FMRFa transcription. Using the Gal4/UAS system we
narrowed Wit's requirement for FMRFa expression to the Tv neurons. As
these neurons accumulate nuclear P-Mad, the results strongly suggest that Wit
is required in the Tv neurons themselves, as opposed to forming part of an
indirect signal relay mechanism. It appears likely that the source of Gbb in
this signaling system is the NHO, as gbb is expressed in the NHO and
replenishing Gbb in the NHO of gbb mutants rescues FMRFa
expression in the Tv neurons. These experiments do not exclude the possibility
that signaling might occur at the cell soma of the Tv neurons in vivo or that
the source of the diffusible ligand could be a different tissue under
physiological conditions. However, the dependence of nuclear P-Mad
accumulation and FMRFa expression in Tv neurons on Dynein-mediated
retrograde transport strongly suggests that signaling is taking place at the
Tv axon terminal. This dependency on Dynein motors is not a general
requirement for FMRFa expression in all neurons because subesophageal
ganglion neurons are not affected by overexpression of dominant-negative Glued
or Dynamitin. Nor is the consequence of disrupting this motor likely to exert
its effect at the level of P-Mad translocation to the nucleus, as nuclear
accumulation of P-Mad in epithelial and mesodermal cells is not effected by
retrograde transport disruption. Only in the nervous system is P-Mad
accumulation specifically affected (McCabe
et al., 2003
), consistent with a role for a retrograde transport
mechanism in moving some component of this signaling pathway from the synapse
to the nucleus.
It is interesting to note that others have recently demonstrated that
misexpressing Gl in the nervous system results in a decrease of quantal
content and the number of synaptic boutons at the larval NMJ
(Eaton et al., 2002
). These
phenotypes are strikingly similar to wit mutants, and are consistent
with the notion that disruption of retrograde transport prevents Bmp signals
from reaching the nucleus. These investigators suggested that the role of
Dynactin is to maintain the presynaptic microtubule cytoskeleton thus
contributing to synapse stability. However, our data and experiments described
elsewhere (McCabe et al.,
2003
) indicate that failure to traffic a Gbb signal from axon to
nucleus also likely contributes to the synaptic defects exhibited by
overexpression of
Gl in motoneurons.
The block of P-Mad accumulation and FMRFa expression upon
disruption of retrograde transport suggests that one of the components of the
signaling pathway has to be transported along the microtubules from the axon
arborizations to the neuron cell body. The normal nuclear accumulation of
P-Mad in epithelial and mesodermal cells in which Dynactin function has been
disrupted makes it unlikely that the signaling block is at the level of
receptor internalization (Penheiter et
al., 2002) or translocation of P-Mad from cytoplasm to the
nucleus. One possibility is that Mad itself could be phosphorylated at the
axon terminal and then transported, either alone or in conjunction with other
components, to the nucleus. However, a second possibility is that an activated
receptor complex is transported back to the cell body. In the case of
Neurotrophins, which constitute the best studied retrograde signaling pathway,
it appears that ligand/activated receptor complexes are internalized through a
clathrin mediated mechanism, and this signaling endosome is then routed to its
appropriate cellular compartment through Dynein mediated retrograde traffic
(Ginty and Segal, 2002
;
Miller and Kaplan, 2001
;
Miller and Kaplan, 2002
;
Yano et al., 2001
). In the
case of Wit, the receptor complex in the signaling endosome would probably
contain the activated type I receptors Tkv and Sax
(Hayes et al., 2002
), and
perhaps also Gbb. Mad phosphorylation could occur in the cytoplasm after
retrograde transport of the signaling endosome. The dependence of Bmp
signaling in the CNS on receptor internalization is supported by analysis of
mutations in the spinster gene. Spinster is a component of the late
endosomes/lysosomes, and mutations in this gene result in enhanced Wit
signaling and synaptic overgrowth, perhaps because of faulty downregulation of
signaling endosomes (Sanyal and Ramaswami,
2002
; Sweeney and Davis,
2002
).
TGFßs as global regulators of synaptic growth and
plasticity
Proper synaptic transmission requires formation, maintenance and pruning or
strengthening of specific synapses in response to developmental or
environmental stimuli. Our observations that Bmps control synaptic activity by
regulating synapse growth as well as the expression of the neuromodulatory
FaRPs, is particularly intriguing in light of another recent report that the
Drosophila Activin pathway also regulates synapse function by
stimulating pruning of mushroom body synapses during the larval to pupal
transition (Zheng et al.,
2003). Whether this involves a retrograde Activin signal is not
clear, but it emphasizes the notion that this family of growth factors appears
to have been recruited multiple times during evolution to regulate different
aspects of synaptic function in invertebrates. Consistent with this view are
previous reports that in Aplysia, TGFß can induce long-term
synaptic facilitation (Chin et al.,
1999
; Zhang et al.,
1997
). Given that various TGFß-type ligands and their
receptors are expressed in specific regions of the adult rodent brain
(Charytoniuk et al., 2000
), it
will be interesting to determine if these proteins also participate in
modulating synaptic function in mammals, particularly in regard to long-term
memory and learning.
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ACKNOWLEDGMENTS |
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Footnotes |
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While this paper was under review, a similar set of findings were described
by Allan et al. (Allan et al.,
2003).
Present address: Dept. of Cell Biology, University of Alabama, Birmingham,
AL35294, USA
Present address: Department of Biological Sciences, Florida Atlantic
University, Boca Raton, FL33431, USA
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